Intra- and extracellular localization of M. genitalium in ME-180 cervical epithelial cells. Cervical ECs (ME-180) were inoculated with log-phase cultures of M. genitalium strain G37 (A) or M2300 (B) to determine whether M. genitalium can invade human reproductive tract ECs. To quantify intracellular M. genitalium loads (solid bar), the inoculum was removed following 3 h incubation for attachment and entry and replaced with medium containing gentamicin (200 ug/mL). The ability for M. genitalium to escape infected ECs (open bar) was quantified from culture supernatants in separate wells processed the same way except, following the 3 h incubation, the inoculum was removed and extracellular M. genitalium organisms were killed with gentamicin (2 h exposure). Infected cells then were washed thoroughly and received fresh medium without gentamicin allowing escaping M. genitalium to survive. Cell fractions or culture supernatants were collected at 48 h following removal of the inoculum for quantification of bacterial loads using a color changing unit (CCU) assay. In every case, significant differences between intracellular and extracellular M. genitalium titers were observed (p < 0.05; Student's t-test). Parallel studies were performed that employed 400 ug/mL gentamicin with similar results (data not shown).